1. Field of the Invention
Coccidiosis is an intestinal disorder of poultry and causes an assortment of problems in the infected host. These problems range from poor feed conversion ratios in light infections to acute death in heavier infections.
Coccidiosis is caused by protozoans belonging to the genus Eimeria. The members of this genus in poultry are E. acervulina, E. tenella, E. maxima, E. necatrix, E. brunetti, E. mitis and E. praecox. Some investigators include E. mivati and E. hagani into the member. All of these species have similar life cycles but display different tissue specificity and pathogenicity. A broiler chicken will be subjected to a great deal of damage by E. acervulina or E. maxima because they parasitize large portions of the small intestine, where food digestion plays a major role.
Coccidiosis can be controlled by the administration of anti-coccidial agents. However, drug resistant strains arise at a frequent rate and the cost of development of new drugs is quite high. In addition, a number of these agents leave residues in the meat, which might give problems on consumer.
Attempts have been made to prevent the disease by vaccinating chickens with live attenuated strains of Eimeria or inactivated parasites. These live attenuated strains such as precocious lines are obtained by inoculating chickens with oocysts of a wild Eimeria species and collecting the very first parasite that are excreted as a result of infection (J Parasitol. 1975, 61: 1083-1090). However, such attenuated live vaccines produce fewer parasites and give an appreciable disease effect to vaccinated chickens. On the other hand, a protection level using the latter (inactivated vaccine) is far from complete. Furthermore, the disadvantage of these vaccines is expensive to be produced because a large-scale production of these vaccines needs a lot of live chickens.
An alternative solution would be to produce, by genetic engineering, the protective antigens of Eimeria parasites. Once developed, these immunogens could be produced cheaply in a prokaryotic or eukaryotic culture system in an unlimited supply and used to vaccinate chickens against coccidiosis.
2. Related Art
Several protective antigen genes of Eimeria have been reported. For examples, Jenkins et al. reported screening using a rabbit serum against the membrane fraction of E. acervulina, and a part of the cDNA encoding a 250-kDa protein in parasite surface (Exp. Parasitol. 1988; 66: 96-107, U.S. Pat. No. 5,122,471). Some Eimeria antigen genes were screened using monoclonal antibodies to Eimeria parasites instead of antisera (U.S. Pat. No. 5,028,694, U.S. Pat. No. 5,279,960, U.S. Pat. No. 5,814,320, U.S. Pat. No. 5,449,768). However, these antigens could elicit only partial protection against Eimeria infection to chickens immunized with a recombinant protein or recombinant virus expressing the antigen (U.S. Pat. No. 5,387,414, U.S. Pat. No. 5,403,581, U.S. Pat. No. 5,602,033, U.S. Pat. No. 6,001,363).
Acquired immune responses are mediated by two different mechanisms, cell-mediated immunity and humoral immunity. The former involves activation of white blood cells such as T-lymphocytes previously sensitized to the immunogen. The latter involves the production of antibodies by lymphoid tissue. Almost all of antigens described above could induce humoral immunity because they were screened by antibodies. However, it is considered that cell-mediated, especially Th1-type immunity plays more important roles in host immune responses against protozoa and bacteria than humoral immunity.
Interestingly, a glycolytic enzyme, GAPDH protein has been shown to be a putative vaccine candidate against Schistosoma japonicum or Schistosoma mansoni infection (Infect. Immun. 1993; 61: 4716-4723, Vaccine 1999; 17: 13-18), and B- and T-cell epitopic regions on GAPDH of S. mansoni have been determined (Vaccine 2000; 18: 2039-2048). Also, GAPDH has been involved as an important immunogenic molecule in several infectious disease models such as African trypanozomiasis and candidasis (J. Infec. Dis. 1995; 172:845-850, Electophoresis 1999; 20: 1001-1010). Moreover, to identify antigen genes that are involved in T-cell-mediated immune response, a genomic library of Brucella abortus has been screened, and the GAPDH has been found as a T- and B-cell reactive protein (J. Med. Microbiol. 2002; 51: 661-671).
Few antigens that involve cell-mediated immunity had been found in Eimeria spp, and it is difficult to identify them by screening only using antibodies against Eimeria parasites.
Moreover, the cDNA library derived from sporocysts or sporozoites, which were not in vivo but in vitro prepared from oocysts, has been used as a screening material in the past. This is because it is very difficult to select only Eimeria cDNA from total cDNA, which were prepared from total mRNA in the intestinal cells of the chicken infected with Eimeria. However, it is thought to be difficult but very important to screen an effective antigen gene that could elicit a cell-mediated immunity using a cDNA library prepared from the intestinal cells of the chicken infected with Eimeria 